The present invention relates to the human genes which encode a specialized group of proteins which promote the growth, maintenance, survival, and functional capabilities of selected populations of neurons.
Neuronotrophic factors (NTFs) are a specialized group of proteins which function to promote the survival, growth, maintenance, and functional capabilities of selected populations of neurons. Recent studies have demonstrated that neuronal death occurs in the nervous systems of vertebrates during certain periods of growth and development. However, the addition of soluble neuronal trophic factors from associated target tissues serves to mitigate this phenomenon of neuronal death. The following citations discuss neuronal trophic factors and their disclosures are hereby incorporated by reference: Chau, R. M. W., et al., Neuronotrophic Factor, 6 Chin. J. Neuroanatomy 129 (1990); Kuno, M., Target Dependence of Motoneuronal Survival: The Current Status, 9 Neurosci. Res. 155 (1990); Bard, Y. A., Trophic Factors and Neuronal Survival, 2 Neuron 1525 (1989); Oppenheim, R. W., The Neurotrophic Theory and Naturally Occurring Motoneuron Death, 12 TINS 252 (1989); Bard, Y. A., What, If Anything, is a Neurotrophic Factor?, 11 TINS 343 (1988); and Thoenen, H., and Edgar, D., Neurotrophic Factors, 229 Science 238 (1985).
In the vertebrate neuromuscular system, the survival of embryonic motoneurons have been found to be dependent upon specific trophic substances derived from the associated developing skeletal muscles. Skeletal muscles have been shown, by both in vivo and in vitro studies, to produce substances which are capable of enhancing the survival and development of motoneurons by preventing the embryonic motoneurons from degeneration and subsequent, natural cellular death. See O""Brian, R. J. and Fischbach, G. D., Isolation of Embryonic Chick Motoneurons and Their Survival In Vitro, 6 J. Neurosci. 3265 (1986); Hollyday, M. and Hamburger, V., Reduction of the Naturally Occurring Motor Neuron Loss by Enlargement of the Periphery, 170 J. Comp. Neurol. 311 (1976), whose disclosures are incorporated herein by reference. Similarly, several investigators have reported that chick and rat skeletal muscles possess certain trophic factors which can prevent the natural cellular death of embryonic motoneurons both in vivo and in vitro. See McManaman, J. L., et al., Purification of a Skeletal Muscle Polypeptide Which Stimulates Choline Acetyltransferase Activity in Cultured Spinal Cord Neurons, 263 J.Biol. Chem. 5890 (1988); Oppenheim, R. W., et al., Reduction of Naturally Occurring Motoneuron Death In Vitro by a Target Derived Neurotrophic Factor, 240 Science 919 (1988); and Smith, R. G., et al., Selective Effects of Skeletal Muscle Extract Fractions on Motoneurons Development In Vivo, 6 J. Neurosci. 439 (1986), whose disclosures are incorporated herein by reference.
In addition, a polypeptide has been isolated from rat skeletal muscle which has been found to selectively enhance the survival of embryonic chick motoneurons in vivo, as well the activity of choline acetyltransferase in these motoneurons. This polypeptide has been named Choline Acetyltransferase Development Factor (CDF) and its biological function has been demonstrated to be different from other trophic factors such as Nerve Growth Factor (NGF), Ciliary Ganglion Neurotrophic Factor (CNTF), Brain-Derived Neurotrophic Factor (BDNF), and Retinal Ganglion Neurotrophic Factor (RGNTF). See Levi-Montalcini, R., xe2x80x9cDevelopmental Neurobiology and the Natural History of Nerve Growth Factor,xe2x80x9d 5 Ann. Rev. Neurosci. 341 (1982); Varon, S., et al., Growth Factors. In: Advances in Neurology, Vol. 47: Functional Recovery in Neurological Disease, Waxman, S. G. (ed.), Raven Press, New York, pp. 493-521 (1988); Barde, Y. A., Trophic Factors and Neuronal Survival, 2 Neuron 1525 (1989); Chau, R. M. W., et al., The Effect of a 30 kD Protein from Tectal Extract of Rat on Cultured Retinal Neurons, 34 Science in China, Series B, 908 (1991), whose disclosures are incorporated herein by reference.
The inventor of the invention disclosed in the instant application, Dr. Raymond Ming Wah Chau, presented results which reported the isolation and characterization of two motoneuronotrophic factors from rat muscle tissue having apparent molecular weights of 35 kD and 22 kD. This data was initially disseminated in 1991 at the World Congress of Cellular and Molecular Biology held in Paris, France. See Chau, R. M. W., et al., Muscle Neuronotrophic Factors Specific for Anterior Horn Motoneurons of Rat Spinal Cord. In: Recent Advances in Cellular and Molecular Biology, Vol. 5, Peeters Press, Leuven, Belgium, pp. 89-94 (1992), the disclosure of which is hereby incorporated by reference. This 35 kD protein has been defined by Dr. Chau as motoneuronotrophic factor 1(MNTF1) and the apparent 22 kD protein as motoneuronotrophic factor 2 (MNTF2). These two trophic factors have been demonstrated in vitro by Dr. Chau to support the growth and/or regeneration of both isolated anterior horn motoneurons and spinal explants of rat lumber spinal cord.
Subsequently, in 1993, Dr. Chau reported the successful cloning of human MNTF1, a protein having an apparent molecular weight of 55 kD, and its associated receptor from a human retinoblastoma cDNA library. See Chau, R. M. W., et al., Cloning of Genes for Muscle-Derived Motoneuronotrophic Factor 1 (MNTF1) and Its Receptor by Monoclonal Antibody Probes, (abstract) 19 Soc. for Neurosci. part 1, 252 (1993), the disclosure of which is hereby incorporated by reference. The cloned human MNTF1 was demonstrated to have biological activity similar to that of the xe2x80x9cnativexe2x80x9d MNTF1 protein in that it supported the in vitro growth of rat anterior horn motoneurons.
Although various biological aspects of MNTF1 have been widely publicized in scientific journals, the DNA and inferred amino acid sequences of the cloned human MNTF1 gene and its associated receptor had not yet been made publicly available by Dr. Chau, nor had these sequences been confirmed by peer-review within the field. Moreover, the cloned human MNTF1, reported by Dr. Chau in 1993, was not in a form which was amenable to being sub-cloned into an appropriate vector, such as an in vitro mammalian expression system. Thus, there remained a need for the human MNTF1 gene to be properly manipulated, sequenced, sub-cloned into an appropriate vector(s), sub-cloned into an appropriate expression system(s) and associated host(s), as well as the isolation and purification of the resulting recombinant human MNTF1 protein for subsequent potential utilization in human therapeutic modalities.
The present invention is directed to a family of motoneuronotrophic factors including MNTF1 and MNTF2, which have been shown to have diagnostic and therapeutic applications in mammals. The present invention is also directed to novel DNA sequences which encode motoneuronotrophic factors, including recombinant human MNTF1-F3 [SEQ ID NO:1] and MNTF1-F6 [SEQ ID NO:2], to vectors which contain these novel DNA sequences, to expression systems and associated hosts which contain these novel DNA sequences, and the novel recombinant human MNTF1-F3 [SEQ ID NO:3] and MNTF1-F6 [SEQ ID NO:4] proteins which are produced by the aforementioned expression systems.
The present invention is also directed to the use of motoneuronotrophic factors for promoting axonal regeneration, for inhibiting the effects of hereditary motoneuron disease, for minimizing or inhibiting the effects of scar tissue formation, and for accelerating wound healing while concomitantly minimizing or inhibiting the effects of scar tissue and keloid formation.